This invention relates to fatty acid desaturases and nucleic acids encoding desaturase proteins. More particularly, the invention relates to nucleic acids encoding delta-12 and delta-15 fatty acid desaturase proteins that affect fatty acid composition in plants, polypeptides produced from such nucleic acids and plants expressing such nucleic acids.
Many breeding studies have been conducted to improve the fatty acid profile of Brassica varieties. Pleines and Freidt, Fat Sci. Technol., 90(5), 167-171 (1988) report plant lines with reduced C18:3 levels (2.5-5.8%) combined with high oleic content (73-79%). Rakow and McGregor, J. Amer. Oil Chem. Soc., 50, 400-403 (Oct. 1973) discuss problems associated with selecting mutants for linoleic and linolenic acids. In Can. J. Plant Sci., 68, 509-511 (Apr. 1988) Stellar summer rape producing seed oil with 3% linolenic acid and 28% linoleic acid is disclosed. Roy and Tarr, Z. Pflanzenzuchtg, 95(3), 201-209 (1985) report transfer of genes through an interspecific cross from Brassica juncea into Brassica napus resulting in a reconstituted line combining high linoleic with low linolenic acid content. Roy and Tarr, Plant Breeding, 98, 89-96 (1987) discuss prospects for development of B. napus LL. having improved linolenic and linolenic acid content. European Patent application 323,753 published Jul. 12, 1989 discloses seeds and oils having greater than 79% oleic acid combined with less than 3.5% linolenic acid. Canvin, Can. J. Botany, 43, 63-69 (1965) discusses the effect of temperature on the fatty acid composition of oils from several seed crops including rapeseed.
Mutations typically are induced with extremely high doses of radiation and/or chemical mutagens (Gaul, H. Radiation Botany (1964) 4:155-232). High dose levels which exceed LD50, and typically reach LD90, led to maximum achievable mutation rates. In mutation breeding of Brassica varieties, high levels of chemical mutagens alone or combined with radiation have induced a limited number of fatty acid mutations (Rakow, G. Z. Pflanzenzuchtg (1973) 69:62-82). The low xcex1-linolenic acid mutation derived from the Rakow mutation breeding program did not have direct commercial application because of low seed yield. The first commercial cultivar using the low xcex1-linolenic acid mutation derived in 1973 was released in 1988 as the variety Stellar (Scarth, R. et al., Can. J. Plant Sci. (1988) 68:509-511). Stellar was 20% lower yielding than commercial cultivars at the time of its release.
Alterations in fatty acid composition of vegetable oils is desirable for meeting specific food and industrial uses. For example, Brassica canola varieties with increased monounsaturate levels (oleic acid) in the seed oil, and products derived from such oil, would improve lipid nutrition. Canola lines which are low in polyunsaturated fatty acids and high in oleic acid tend to have higher oxidative stability, which is a useful trait for the retail food industry. Useful traits of vegetable oils for industrial uses like lubrication fluids include desirable low temperature behavior such as low pour point and low cloud point along with very high oxidative stability.
Delta-12 fatty acid desaturase (also known as oleic desaturase) is involved in the enzymatic conversion of oleic acid to linoleic acid. Delta-15 fatty acid desaturase (also known as linoleic acid desaturase) is involved in the enzymatic conversion of linoleic acid to xcex1-linolenic acid. A microsomal delta-12 desaturase has been cloned and characterized using T-DNA tagging. Okuley, et al., Plant Cell 6:147-158 (1994). The nucleotide sequences of higher plant genes encoding microsomal delta-12 fatty acid desaturase are described in Lightner et al., WO94/11516. Sequences of higher plant genes encoding microsomal and plastid delta-15 fatty acid desaturases are disclosed in Yadav, N., et al., Plant Physiol., 103:467-476 (1993), WO 93/11245 and Arondel, V. et al., Science, 258:1353-1355 (1992).
Triacylglycerols containing fatty acids with heterogenous chain lengths and with high monounsaturate levels can provide useful traits for industrial purposes. Plants with fatty acid compositions that have high monounsaturate levels and heterogenous chain lengths would provide a source of industrial oils for uses such as lubrication.
In one aspect, the invention features a Brassica plant, and progeny thereof, producing seeds having a long chain monounsaturated fatty acid content of at least about 82% and an erucic acid content of at least about 15% based on total fatty acid composition. The oleic acid and eicosenoic acid content of the seeds is at least about 37% and at least about 14%, based on total fatty acid composition, respectively. The saturated fatty acid content of such seeds is less than 7% and the polyunsaturated fatty acid content is less than about 11%.
In some embodiments, the plants have a monounsaturated fatty acid content of from about 85% to about 90% and an erucic acid content of at least about 15% based on total fatty acid composition. In such plants, the oleic acid content can be at least about 42% and in particular, from about 47% to about 56% based on total fatty acid composition. The erucic acid content is from about 17% to about 31%, and the eicosenoic acid content is from about 15% to about 21%.
The invention also features a Brassica seed oil having a long chain monounsaturated fatty acid content of at least about 82% and an erucic acid content of at least about 15% based on total fatty acid composition. Such oils can have an oleic acid and eicosenoic acid content of at least about 14% and 37%, respectively, based on total fatty acid composition. The saturated fatty acid content is less than about 7%. The polyunsaturated fatty acid content is less than about 11% and in particular embodiments, less than 9%, based on total fatty acid composition.
In some embodiments, the Brassica seed oil contains a long chain monounsaturated fatty acid content of from about 85% to about 90%. In such oils, the oleic acid content is at least about 42%, and in particular embodiments, is from about 47% to about 56%, based on total fatty acid composition. The erucic acid and eicosenoic acid content is from about 17% to about 31% and from about 15% to about 21%, respectively, based on total fatty acid composition.
The invention also features a method of producing plants having a long chain monounsaturated fatty acid content of at least about 82% and an erucic acid content of at least about 15%, based on total fatty acid composition. The methods include crossing a first plant line with a second plant line and selecting progeny with the desired fatty acid composition. The first plant line has an erucic acid content of at least about 45%. The second plant line has an oleic acid content of at least about 84%.
SEQ ID NO:1 shows a hypothetical DNA sequence of a Brassica Fad2 gene. SEQ ID NO:2 is the deduced amino acid sequence of SEQ ID NO:1.
SEQ ID NO:3 shows a hypothetical DNA sequence of a Brassica Fad2 gene having a mutation at nucleotide 316. SEQ ID NO:4 is the deduced amino acid sequence of SEQ ID NO:3.
SEQ ID NO:5 shows a hypothetical DNA sequence of a Brassica Fad2 gene. SEQ ID NO:6 is the deduced amino acid sequence of SEQ ID NO:5.
SEQ ID NO:7 shows a hypothetical DNA sequence of a Brassica Fad2 gene having a mutation at nucleotide 515. SEQ ID NO:8 is the deduced amino acid sequence of SEQ ID NO:7.
SEQ ID NO:9 shows the DNA sequence for the coding region of a wild type Brassica Fad2-D gene. SEQ ID NO:10 is the deduced amino acid sequence for SEQ ID NO:9.
SEQ ID NO:1 shows the DNA sequence for the coding region of the IMC 129 mutant Brassica Fad2-D gene. SEQ ID NO:12 is the deduced amino acid sequence for SEQ ID NO:11.
SEQ ID NO:13 shows the DNA sequence for the coding region of a wild type Brassica Fad2-F gene. SEQ ID NO:14 is the deduced amino acid sequence for SEQ ID NO:13.
SEQ ID NO:15 shows the DNA sequence for the coding region of the Q508 mutant Brassica Fad2-F gene. SEQ ID NO:16 is the deduced amino acid sequence for SEQ ID NO:15.
SEQ ID NO:17 shows the DNA sequence for the coding region of the Q4275 mutant Brassica Fad2-F gene. SEQ ID NO:18 is the deduced amino acid sequence for SEQ ID NO:17.